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PubMed1999

Ubiquitous expression and embryonic requirement for RNA polymerase II coactivator subunit Srb7 in mice.

Mammalian RNA polymerase II complexes and coactivators containing homologs of yeast Srb/Med proteins have been isolated recently from tissue culture cells. The yeast Srb/Med complex is involved in global gene expression and is essential, but it is not yet known if its mammalian counterparts are broadly expressed in tissues or if they are essential. We have isolated the murine gene encoding Srb7, an Srb/Med complex protein whose sequence and function is highly conserved between yeast and humans. The mouse Srb7 gene is single copy, and Northern analysis showed that it is expressed in all tissues examined. Disruption of the gene in embryonic stem cells revealed that it is essential for cell viability and murine embryonic development. These results, together with evidence that murine Srb7 is associated exclusively with high molecular weight forms of RNA polymerase II in extracts, suggest that Srb7-containing polymerase complexes occur in most tissues and have essential roles in expression of protein coding genes.

Amino Acid Sequence↗

PubMed2003

Identification by subtractive hybridization of a novel insertion element specific for two widespread Burkholderia cepacia genomovar III strains.

Species of the Burkholderia cepacia complex cause chronic and life-threatening infections in persons with cystic fibrosis. Epidemic strains infect multiple patients, reside primarily in genomovar III, and have an apparent enhanced capacity for human infection and/or interpatient transmission. By using subtractive hybridization, a novel insertion element, designated IS1363, was identified in epidemic strain PHDC, known to infect many cystic fibrosis patients in the mid-Atlantic region of the United States. IS1363 was also found in most isolates of the ET12 lineage, responsible for infecting large numbers of patients in Ontario, Canada, and the United Kingdom. Southern blot analysis demonstrated that whereas multiple copies of IS1363 were present in strain PHDC, only one copy was present in ET12 isolates. IS1363 was used to probe a collection of 943 B. cepacia complex isolates, representing all nine genomovars, recovered from 761 cystic fibrosis patients or the natural environment. IS1363 was not found in other genomovar III strains and, with the exception of B. ambifaria, was absent from other B. cepacia complex species. Genotyping analyses of all IS1363-positive isolates demonstrated that strain PHDC was more widely distributed in the United States than previously appreciated; 212 cystic fibrosis patients in 24 states were identified as being infected with PHDC.

Bacterial Typing Techniques↗

PubMed2026

MarkerMatch: a proximity-based probe-matching algorithm for joint analysis of copy-number variants from different genotyping arrays.

MOTIVATION: Copy-number variants (CNVs) are a form of genetic structural variation with increasing importance in complex human disorders. Both DNA sequencing and microarray data can be used to detect CNVs, which can be used in genetic association tests. Unlike genotypes, CNV detection in microarrays requires the use of observed intensity signals at each probe, which limits the imputability for analyses that span multiple array types. Thus far, a consensus set of probes (those present on all arrays) has been used to circumvent the problem of differing array-specific sensitivities. This has led to excessive reduction in overall sensitivity since arrays can have an undesirably low probe overlap. To overcome this limitation, we developed MarkerMatch, a proximity-based algorithm that matches probes across different genotyping microarrays to maximize the number of probes considered in the CNV calling algorithm, thereby increasing the resolution and sensitivity while preserving precision. RESULTS: By analyzing CNV calls from 4906 individuals genotyped across three different arrays, we show that the MarkerMatch approach improves sensitivity by increasing the density of probes available for CNV calling while maintaining precision or improving it relative to the current practice (e.g. use of consensus probes only). We further demonstrate that MarkerMatch matches the CNV detection from current practice in terms of F1 score and PPV for larger CNVs. We also optimize MarkerMatch parameters, DMAX and Method, and find an optimal DMAX setting at 10 kb, with no clear optimal candidate based on Method, indicating that parameters for this metric should be determined on a use case basis. AVAILABILITY: The R package for MarkerMatch is available at: https://github.com/FranjoIM/MarkerMatch. The code used for analysis and implementation is available at: https://doi.org/10.5281/zenodo.18460979. The live notebook is available at https://fivankovic.notion.site/2026-markermatch.

DNA Copy Number Variations↗

PubMed1968

Visual perception and xerography.

An electrostatic copying machine was used to model the perception of simultaneous brightness contrast. Such a model may assist the study of sensory inhibition by permitting the study of complex situations as they are transformed by rules similar to those at work in neural integration.

Copying Processes↗

PubMed2001

A complex of the bacteriophage T7 primase-helicase and DNA polymerase directs primer utilization.

The lagging strand of the replication fork is initially copied as short Okazaki fragments produced by the coupled activities of two template-dependent enzymes, a primase that synthesizes RNA primers and a DNA polymerase that elongates them. Gene 4 of bacteriophage T7 encodes a bifunctional primase-helicase that assembles into a ring-shaped hexamer with both DNA unwinding and primer synthesis activities. The primase is also required for the utilization of RNA primers by T7 DNA polymerase. It is not known how many subunits of the primase-helicase hexamer participate directly in the priming of DNA synthesis. In order to determine the minimal requirements for RNA primer utilization by T7 DNA polymerase, we created an altered gene 4 protein that does not form functional hexamers and consequently lacks detectable DNA unwinding activity. Remarkably, this monomeric primase readily primes DNA synthesis by T7 DNA polymerase on single-stranded templates. The monomeric gene 4 protein forms a specific and stable complex with T7 DNA polymerase and thereby delivers the RNA primer to the polymerase for the onset of DNA synthesis. These results show that a single subunit of the primase-helicase hexamer contains all of the residues required for primer synthesis and for utilization of primers by T7 DNA polymerase.

Amino Acid Sequence↗

PubMed1997

A Drosophila Tpr protein homolog is localized both in the extrachromosomal channel network and to nuclear pore complexes.

Here we report structural, molecular, and biochemical characterizations of Bx34, a Drosophila melanogaster nuclear coiled-coil protein which is localized to extrachromosomal and extranucleolar spaces in the nuclear interior and which is homologous to the mammalian nuclear pore complex protein Tpr. In the nuclear interior, Bx34 is excluded from chromosomes and the nucleolus and generally localizes to regions between these structures and the nuclear periphery. This distribution matches the 'extrachromosomal channel network' described previously. In the nuclear periphery, Bx34 localizes on or near nuclear pore complexes. Biochemically, Bx34 isolates exclusively with the nuclear matrix fraction. The Bx34 cDNA sequence predicts a large protein (262 kDa) with two distinct structural domains. The Bx34 N-terminal 70% (180 kDa) is predicted to form an extended region of coiled-coil, while the C-terminal 30% (82 kDa) is predicted to be unstructured and acidic. Bx34 shows moderate sequence identity over its entire length to the mammalian nuclear pore complex protein 'Tpr' (28% amino acid identity and 50% similarity). Furthermore, several of the sequence motifs and biochemical similarities between Bx34 and Tpr are sufficiently striking that it is likely that Bx34 and Tpr are functionally related. The Bx34 gene exists in a single copy in region 48C of chromosome 2R. The localization of coiled-coil Bx34 to both the nuclear interior and nuclear pore complexes and its sequence similarity to a known nuclear pore complex protein leads to speculations about a role for Bx34 in nucleo-cytoplasmic transport which we can test using molecular genetic approaches.

Amino Acid Sequence↗

PubMed2026

Long-read based detection of large copy number variants with potential functional significance using the ContextSV structural variant caller.

Long-read sequencing enables improved detection of structural variants (SVs) in the human genome due to its substantially increased read lengths. However, currently widely used long-read SV callers primarily rely on alignment-based evidence, limiting their ability to detect large and complex SVs and potentially missing disease-relevant events. To address these limitations, we developed ContextSV, a framework that integrates alignment evidence with copy number predictions derived from sequencing coverage and single-nucleotide variant allele frequencies to improve SV detection, particularly for large copy number variants (CNVs). We additionally developed ContextScore, a machine learning-based classification model to assign SV confidence scores based on genomic context features and integrated it within ContextSV. Through benchmarking analyses on both simulated and real datasets, we demonstrate that ContextSV improves detection of large CNVs and inversions that may be missed by existing long-read SV callers. We further illustrate its utility by identifying and experimentally validating multiple large SVs in the KOLF2.1J reference stem cell line that were not detected by other methods. Collectively, our results demonstrate that ContextSV serves as a valuable complement to existing long-read SV detection approaches by improving sensitivity for large and clinically relevant SVs.

Humans↗

PubMed2004

Good copy, bad copy: choosing animal models for HLA-linked diseases.

Recent large-scale sequencing and comparative analyses of the major histocompatibility complex (Mhc) provide a novel view of this long-studied region. The main insight is that even though Mhcs are defined by the presence of the Mhc class I and II genes, the regions encoding class I/II histocompatibility antigens are the least conserved among the species; hence the difficulty of modeling the human class I/II-linked diseases. Fortunately, the majority of the genes in the Mhc, the non-class I/II genes, are conserved among the investigated mammals. The full set of Mhc genes in their evolutionary context presents new possibilities to study Mhc-linked diseases by allowing systematic evaluation of the various experimental animals and approaches.

Alleles↗

PubMed2003

Type I photosynthetic reaction centres: structure and function.

We review recent advances in the study of the photosystem I reaction centre, following the determination of a spectacular 2.5 A resolution crystal structure for this complex of Synechococcus elongatus. Photosystem I is proving different to type II reaction centres in structure and organization, and the mechanism of transmembrane electron transfer, and is providing insights into the control of function in reaction centres that operate at very low redox potentials. The photosystem I complex of oxygenic organisms has a counterpart in non-oxygenic bacteria, the strictly anaerobic phototrophic green sulphur bacteria and heliobacteria. The most distinctive feature of these type I reaction centres is that they contain two copies of a large core polypeptide (i.e. a homodimer), rather than a heterodimeric arrangement of two related, but different, polypeptides as in the photosystem I complex. To compare the structural organization of the two forms of type I reaction centre, we have modelled the structure of the central region of the reaction centre from green sulphur bacteria, using sequence alignments and the structural coordinates of the S. elongatus Photosystem I complex. The outcome of these modelling studies is described, concentrating on regions of the type I reaction centre where important structure-function relationships have been demonstrated or inferred.

Amino Acid Sequence↗

PubMed2002

Molecular evidence to support a proposal to reserve the designation Mycobacterium avium subsp. avium for bird-type isolates and 'M. avium subsp. hominissuis' for the human/porcine type of M. avium.

In an attempt to clarify the taxonomy of the Mycobacterium avium complex, the relationship between IS1245 RFLP, growth temperature, 16S rDNA signature sequences and the 16S-23S rDNA internally transcribed spacer (ITS) of 160 M. avium-complex isolates from different sources was investigated. All 70 isolates identified as M. avium by INNO-LiPA MYCOBACTERIA (Innogenetics, Belgium), a DNA probe test that targets the ITS, and by 16S rDNA analysis carried multiple copies of IS1245. Three isolates with multiple copies of IS1245 were identified by 16S rDNA analysis as Mycobacterium intracellulare and by LiPA as M. intracellulare (n = 1) and M. avium-intracellulare complex (n = 2). A dichotomy among the M. avium isolates was found on the basis of a C and a G signature nucleotide at position 228 of the 16S-23S rDNA spacer sequence, and this grouping was largely confirmed on the basis of similarities in IS1245 RFLPs. Strains with the characteristic three-band IS1245 'bird-type', as well as M. avium subsp. silvaticum or 'wood-pigeon' strains, invariably contained the C signature. A third characteristic that separated the M. avium bird-type isolates from M. avium isolates from humans and other mammals was growth-temperature tolerance: in contrast to bird isolates, human/porcine isolates grew at 24 and 45 degrees C. Based on differences in IS1245 RFLP, 16S-23S rDNA ITS and growth temperature, M. avium isolates originating from birds should be considered as a separate, evolutionarily conserved taxon. Because all M. avium isolates from birds are invariably of this type, the designation M. avium subsp. avium should be reserved for these bird-type strains. For clarity in the epidemiology of M. avium-related disease, isolates from humans and pigs with multibanded IS1245 RFLPs merit a separate designation. The designation 'M. avium subsp. hominissuis' is suggested for this group of bacteria.

Animals↗

PubMed1995

Molecular analysis of Caenorhabditis elegans tcp-1, a gene encoding a chaperonin protein.

A Caenorhabditis elegans (Ce) homologue to the eukaryotic tcp-1 gene (encoding t-complex polypeptide-1) has been mapped, isolated and sequenced. Ce tcp-1 is a single-copy gene located on chromosome II. Nucleotide sequence analysis of the gene reveals the presence of four introns in the coding region and repetitive elements upstream from the start codon. The predicted Ce TCP-1 protein displays more than 60% amino-acid sequence identity to other eukaryotic TCP-1, suggesting a common origin and function for these proteins. The primary tcp-1 transcript undergoes trans-splicing to the spliced leader SL1 RNA, in addition to cis-splicing, to yield a single mRNA species of 1.9 kb. Northern blot analysis shows that unlike the evolutionarily related Hsp60 chaperonin genes, tcp-1 is not upregulated at elevated temperatures, but instead appears to be down-regulated. Additionally, the overall level of the tcp-1 transcript is approximately constant throughout the development of the nematode. The Ce chaperonin-containing TCP-1 (CCT) was identified. A protein extract made from Ce embryos was subjected to sucrose gradient fractionation and ATP-agarose chromatography. Western blot analysis of the purified protein fractions, using anti-mouse TCP-1 monoclonal antibody and antibodies raised against Ce TCP-1, reveals that Ce TCP-1 is a 57-kDa protein subunit of a high-molecular-mass complex capable of binding ATP.

Amino Acid Sequence↗

PubMed1996

Isolation of the mRNA-capping enzyme and ferric-reductase-related genes from Candida albicans.

The mRNA-capping enzyme (mRNA 5'-guanylyltransferase) gene was cloned from a Candida albicans genomic DNA library by functional complementation of a Saccharomyces cerevisiae ceg1 delta null mutation. This gene, referred to as CGT1 (C. albicans guanylyltransferase 1), can encode a 52 kDa protein that is highly homologous to S. cerevisiae Ceg1p. CGT1 in a single-copy plasmid complemented the lethality of the S. cerevisiae ceg1 delta null mutation and, like S. cerevisiae Ceg1p, bacterially expressed Cgt1p was able to form a stable complex with the GMP moiety of GTP and to synthesize the cap structure in vitro, demonstrating that CGT1 is the C. albicans mRNA 5'-guanylyltransferase gene. CGT1 seemed to exist as a single copy in the C. albicans genome and was actively transcribed into mRNA. Another ORF was found in an opposite strand very close to the CGT1 locus. This gene shared significant sequence homology with S. cerevisiae FRE1, the gene encoding ferric reductase, and therefore was designated CFL1 (C. albicans ferric-reductase-like gene 1). Despite its sequence homology with S. cerevisiae FRE1, CFL1 mRNA was not induced by iron deprivation, and CFL1 did not complement the slow growth of a S. cerevisiae fre1 delta null mutant in the absence of iron, suggesting that CFL1 is functionally distinct from S. cerevisiae FRE1.

Amino Acid Sequence↗

PubMed1994

Stable fiber-forming and nonfiber-forming chaperone-subunit complexes in pilus biogenesis.

The P pilus is a composite fiber consisting of a thin adhesive tip fibrillum joined to the pilus rod that mediates specific adherence of uropathogenic Escherichia coli to human uroepithelial cells via the PapG tip adhesin. P pilus assembly depends upon the periplasmic chaperone PapD. The interaction of PapD with different pilus subunits was investigated to gain further insight into pilus assembly. PapA, the major subunit of the pilus rod, formed two periplasmic complexes (DA2 and DA) with PapD. PapK, an adaptor protein that joins the tip fibrillum to the pilus rod, formed only one complex with PapD (DK). Only "fiber forming" or homopolymeric subunits, PapA in the rod and PapE in the tip fibrillum, were able to form subunit-subunit interactions in the periplasm. Subunits that are present in single or low copy in the pilus (PapK and PapG) did not form periplasmic intersubunit interactions. A pulse-chase analysis revealed that a chaperone-PapA complex is a true periplasmic intermediate in pilus assembly.

Bacterial Proteins↗

PubMed2005

Topoisomerase II suppresses the temperature sensitivity of Saccharomyces cerevisiae pds5 mutants, but not the defect in sister chromatid cohesion.

Sister chromatid cohesion enables chromosomes to achieve bipolar attachment to the mitotic spindle and its dissolution is required for chromosome segregation. The cohesin complex serves as the primary molecular glue responsible for cohesion. Pds5p binds to the same chromosomal loci as the cohesin complex but plays a distinct role as a regulator of cohesion maintenance. Catenation between sister chromatids must also be removed by Topoisomerase II (Top2p) enzymatic activity to enable chromosome segregation. We identified TOP2 as a high-copy suppressor of the temperature sensitivity of pds5 mutants. TOP2 suppression is specific for pds5 mutants as it does not suppress mutants in the cohesin complex. TOP2 suppresses mini-chromosome loss in pds5 mutants indicating that it rescues a chromosome segregation defect. Surprisingly, TOP2 overexpression fails to suppress the cohesion defect of pds5 mutants, suggesting that it suppresses an additional and as yet uncharacterized defect in pds5 mutants that is essential for viability. A catalytically dead TOP2 allele suppresses pds5 temperature sensitivity, suggesting that suppression is unrelated to Top2p enzymatic function. Consistent with this idea, when the pds5 mutant is combined with the top2-4 mutant, which accumulates DNA catenanes due to defective enzymatic activity, the double mutants exhibit synthetic sickness indicating that increased catenation is toxic to pds5 cells. Our results suggest that Pds5p and Top2p cooperate to promote proper chromosome segregation by a mechanism unrelated to either cohesion or catenation/decatenation.

Alleles↗

PubMed1984

[Isolation and characteristics of poly(A+) RNA in the lactating bovine udder].

Total cellular poly(A+)-RNA was isolated from a lactating cow mammary gland. The poly(A+)-RNA molecules exhibit a heterogeneous distribution from 500 to 5000 nucleotides (average size--1600 nucleotides) and are made up of three main fractions (1550, 950 and 600 nucleotides) possessing a high template activity during translation in vitro. Optimal conditions for poly(A+)-RNA translation in a cell-free protein-synthesizing system from wheat embryos were elaborated. Immunochemical analysis of translation products revealed that 30% of the synthesized polypeptides are precipitated by immunoglobulins against cow milk proteins. Using hybridization with homologous cDNA, the kinetic complexity and heterogeneity of total cellular poly(A+)-RNA were investigated. This population was shown to consist of four classes differing in the diversity of their nucleotide sequences and the number of copies per cell. The total amount of the poly(A+)-RNA species in the cells of a lactating cow mammary gland is 9200, i.e., 0.46% of the genome complexity.

Animals↗

PubMed2026

Pleomorphic Liposarcoma: Comprehensive Genomic Analysis of 39 Cases With Comparison to Other Genomically Complex Sarcomas.

Pleomorphic liposarcoma (PLPS) is an aggressive high-grade sarcoma that often shows diverse morphological features and can mimic high-grade undifferentiated pleomorphic sarcoma (UPS)/spindle cell sarcoma or myxofibrosarcoma (MFS), especially when pleomorphic lipoblasts are sparse. The molecular profile of PLPS is distinct from well differentiated/dedifferentiated liposarcoma and myxoid liposarcoma. In this study, we investigate 39 cases of PLPS by comprehensive genomic profiling, occurring in 32 patients with available molecular data. Cases were reviewed and morphologic parameters-lipoblastic component, UPS-like, and MFS-like areas were estimated. The genomic findings were collected and compared to UPS and MFS groups studied using the same platform. The cohort included 15 females and 17 males, with a median age of 56.5 (range, 34-78). The lower extremity (n = 17) was the most common site involved, followed by upper extremity (n = 5) and pelvis (n = 5). UPS-like and MFS-like patterns were the most common morphologic variants, ranging from 15% to 95% and 20% to 90%, respectively. TP53 (87%) and RB1 (51%) mutations and copy number alterations were the most common alterations seen, followed by ATRX (36%). Compared to UPS and MFS, TP53 and RB1 gene alterations were significantly more common in PLPS. Conversely, CDKN2A/B deletions were infrequent in PLPS. Survival analysis showed that MYC amplification was associated with significantly shorter overall survival in PLPS. Among histologic variants, CYSLTR2 alterations were found to be highest in cases with predominantly pleomorphic lipoblasts; additionally, strong correlations were found between gene alteration frequencies of MFS and MFS-like PLPS, and between UPS and UPS-like PLPS. RB1 allele-specific copy number analysis showed loss of heterozygosity in 82% of cases. Our cohort of PLPS showed a complex molecular landscape with distinct genetic alterations, histologic correlations, and clinical outcomes, highlighting its unique position among genomically complex sarcomas and providing insights that may inform future diagnostic and therapeutic approaches.

Humans↗

PubMed1984

Organ-specific nuclear RNAs in tobacco.

We investigated the developmental regulation of nuclear RNA sequences in tobacco vegetative (leaf, root, stem) and floral (petal, ovary, anther) organ systems using RNA-excess X single-copy DNA hybridization reactions. We found that 18% of the single-copy DNA, equivalent to 1.1 X 10(5) kilobases (kb) of diverse transcripts, is represented in the nuclear RNA of each organ. Each nuclear RNA population has both shared and organ-specific sequences. Depending upon the nuclear RNA, 10-40% of the complexity, or 1.1-4.4 X 10(4) kb of diverse sequence, is organ-specific. Collectively, at least 45% of the single-copy DNA, or 3 X 10(5) kb, is represented in the nuclear RNA of the entire plant. Hybridization experiments with polysomal RNA showed that organ-specific mRNAs are present in both the unique and shared nuclear RNA subsets. Together, our results show that tobacco nuclear RNA sequences are under striking developmental control and that both transcriptional and post-transcriptional processes play a role in regulating plant gene expression.

Base Sequence↗

PubMed1998

The partitioning activity of the RK2 central control region requires only incC, korB and KorB-binding site O(B)3 but other KorB-binding sites form destabilizing complexes in the absence of O(B)3.

The sector of the genome of broad-host-range IncP plasmid RK2 from kb coordinate 54.0 to 60.0 confers an active partitioning phenotype, increasing the segregational stability of low-copy-number unstable plasmids. This Par region encodes the central control operon (korA, incC, korB, korF and korG) and the associated genes kfrA, upf54.8 and upf54.4. Each ORF in this region was knocked out in turn and it was shown that only incC and korB are needed for the stability phenotype. incC encodes two polypeptides from alternative translational starts. A deletion of the start of the operon showed that only IncC2, the shorter product, is essential for partitioning. Directed mutation or deletion was used to inactivate in turn each of the three KorB-binding sites (O(B)s) which were candidate cis-acting sequences needed for stability. Only inactivation of O(B)3, which lies between upf54.4 and upf54.8, resulted in an increased rate of segregational loss. However, the rate of loss was significantly higher than the rate of loss of the test plasmid carrying none of this RK2 Par region. Either inactivation of korB or deletion of O(B)1 from this O(B)3 mutant resulted in restoration of the loss rate to that expected for the unstable test plasmid alone. Thus KorB can act on O(B)1 to create a complex that either inhibits replication or reduces the effective plasmid copy number, perhaps by promoting pairing between plasmid molecules. This implies that RK2 goes through a cycle of pairing and separation, akin to the mitotic cycle of eukaryotic chromosomes.

Bacterial Proteins↗